ABSTRACT While the importance of chemical modifications of DNA and proteins is well established, relatively little is known about how the post-transcriptional modification of mRNA transcripts affects their function. The most common modified base seen on cellular mRNAs in mammals is N6-methyladenosine (m6A), and recent data demonstrate that the total loss of m6A formation can have severe deleterious consequences, for example blocking the differentiation of pluripotent stem cells. However, how m6A regulates mRNA expression remains to be established. Moreover, previous work has revealed that several viruses encode mRNAs that undergo m6A modification suggesting that such modifications likely exert a positive effect on virus replication. This grant proposal seeks to define the sites of m6A modification on HIV-1 transcripts and to determine how these modifications affect HIV-1 gene expression and replication. Using the PAR-CLIP technique, we have identified several specific binding sites for the human YTHDF m6A reader proteins on the HIV-1 genome that are also bound in vitro by an m6A-specific antibody. Of interest, these m6A editing sites are conserved across a wide range of HIV-1 isolates. We have demonstrated that these viral m6A sites are maintained in indicator constructs containing segments of the HIV-1 genome and have observed that mutagenesis of these m6A sites results in a significant reduction in mRNA expression. Consistent with the hypothesis that m6A editing positively regulates HIV-1 gene expression, we have observed that overexpression of the human YTHDF2 reader protein in human CD4+ T cells enhances HIV-1 replication while knock out of the YTHDF2 gene by genome editing inhibits HIV-1 replication. This grant first aims to define the locations of m6A modifications on the HIV-1 genome at single nucleotide resolution and to then quantify the degree of editing at each site. We will then use targeted viral mutagenesis to determine precisely how specific m6A modifications affect HIV-1 gene expression and replication. In parallel, we will examine how the overexpression, in CD4+ T cells, of key cellular proteins involved in ?writing?, ?reading? and ?erasing? m6A marks, or their elimination by genome editing using CRISPR/Cas, affects HIV-1 replication. Finally, we intend to extend this analysis to the experimentally tractable animal retrovirus murine leukemia virus in order to determine whether m6A editing represents a general strategy to promote retroviral gene expression and replication.